Apoptosis (or programmed cell death) plays critical roles in normal development and tissue homeostasis in multi-cellular organisms. Impaired apoptosis is both critical in the development of a variety of human diseases and a major barrier to effective treatment. The Bcl-2 family of proteins constitutes a crucial checkpoint in the apoptosis pathway. Bax is a pro-apoptotic member of the Bcl-2 family that controls the mitochondrion-mediated cell death pathway. In normal healthy cells, Bax locates in the cytosol and is maintained in an inactive state. Upon apoptotic stimulation, Bax undergoes conformational changes, and migrates from the cytosol to mitochondria. With insertion of its C-terminal transmembrane domains into the outer membrane of mitochondria, Bax disrupts mitochondrial membrane potential, and induces release of pro-apoptotic proteins (such as cytochrome c) from mitochondria to cytosol. The released pro-apoptotic proteins trigger cellular apoptosis. What factors maintain Bax in its inactive conformation in the cytosol of healthy cells is not fully understood. We hypothesize that novel cell death regulatory proteins that interact with Bax and keep Bax in check in the cytosol of healthy cells may exist. Our preliminary studies showed that in addition to the majority of monomeric Bax, a small portion of Bax was associated with high molecular weight protein complexes in the cytosol of healthy cells. We propose to use novel proteomic method to systematically identify the proteins that associate with Bax in the cytosol of healthy cells (Specific Aim 1). After validation of the interaction between Bax and the identified proteins, the expression of the identified proteins in cells will be manipulated by RNAi or overexpression, and their effects on Bax conformational changes, Bax translocation to mitochondria, and apoptosis will be examined in cells. The identifications of novel Bax interacting, death regulatory proteins are critical for understanding the molecular mechanisms of Bax activation in apoptosis. Given the important roles of Bax in the development and treatment of human diseases, the results may provide valuable information for designing new strategies to prevent and treat human diseases including cancer an neurodegenerative disorders. PUBLIC HEALTH RELEVANCE: The objective of this project is to use novel quantitative proteomic method to systematically identify Bax interacting proteins that may play critical roles in keeping Bax in check in the cytosol of healthy cells. The identifications of novel Bax interacting, death regulatory proteins are critical for understanding the molecular mechanisms of Bax activation in apoptosis. Molecular and cell biological techniques will be used to assess the biological functions of the identified proteins.